Neurocristopathies: Enigmatic Appearances of Neural Crest Cell-derived Abnormalities.

The neural crest is an important transient structure that develops during embryogenesis in vertebrates. Neural crest cells are multipotent progenitor cells that migrate and develop into a diverse range of cells and tissues throughout the body. Although neural crest cells originate from the ectoderm, they can differentiate into mesodermal-type or endodermal-type cells and tissues. Some of these tissues include the peripheral, autonomic, and enteric nervous systems; chromaffin cells of the adrenal medulla; smooth muscles of the intracranial blood vessels; melanocytes of the skin; cartilage and bones of the face; and parafollicular cells of the thyroid gland. Neurocristopathies are a group of diseases caused by the abnormal generation, migration, or differentiation of neural crest cells. They often involve multiple organ systems in a single person, are often familial, and can be associated with the development of neoplasms. As understanding of the neural crest has advanced, many seemingly disparate diseases, such Treacher Collins syndrome, 22q11.2 deletion syndrome, Hirschsprung disease, neuroblastoma, neurocutaneous melanocytosis, and neurofibromatosis, have come to be recognized as neurocristopathies. Neurocristopathies can be divided into three main categories: dysgenetic malformations, neoplasms, and combined dysgenetic and neoplastic syndromes. In this article, neural crest development, as well as several associated dysgenetic, neoplastic, and combined neurocristopathies, are reviewed. Neurocristopathies often have clinical manifestations in multiple organ systems, and radiologists are positioned to have significant roles in the initial diagnosis of these disorders, evaluation of subclinical associated lesions, creation of treatment plans, and patient follow-up. Online supplemental material is available for this article. ©RSNA, 2019.

Altered differentiation of enteric neural crest-derived cells from endothelin receptor-B null mouse model of Hirschsprung's disease.

PURPOSE: Hirschsprung's disease (HD) is caused by a failure of enteric neural crest-derived cells (ENCC) to colonize the bowel, resulting in an absence of the enteric nervous system (ENS). Previously, we developed a Sox10 transgenic version of the Endothelin receptor-B (Ednrb) mouse to visualize ENCC with the green fluorescent protein, Venus. The aim of this study was to isolate Sox10-Venus+ cells, which are differentiated neurons and glial cells in the ENS, and analyze these cells using Sox10-Venus mice gut. METHODS: The mid-and hindgut of Sox10-Venus+/Ednrb +/+ and Sox10-Venus+/Ednrb -/- at E13.5 and E15.5 were dissected and cells were dissociated. Sox10-Venus+ cells were then isolated. Expression of PGP9.5 and GFAP were evaluated neurospheres using laser scanning microscopy. RESULTS: 7 days after incubation, Sox10-Venus+ cells colonized the neurosphere. There were no significant differences in PGP9.5 expressions on E13.5 and E15.5. GFAP was significantly increased in HD compared to controls on E15.5 (P < 0.05). CONCLUSIONS: Our results suggest increased glial differentiation causes an imbalance in ENCC lineages, leading to a disruption of normal ENS development in this HD model. Isolation of ENCC provides an opportunity to investigate the ENS with purity and might be a useful tool for modeling cell therapy approaches to HD.

Regulators of gene expression in Enteric Neural Crest Cells are putative Hirschsprung disease genes.

The enteric nervous system (ENS) is required for peristalsis of the gut and is derived from Enteric Neural Crest Cells (ENCCs). During ENS development, the RET receptor tyrosine kinase plays a critical role in the proliferation and survival of ENCCs, their migration along the developing gut, and differentiation into enteric neurons. Mutations in RET and its ligand GDNF cause Hirschsprung disease (HSCR), a complex genetic disorder in which ENCCs fail to colonize variable lengths of the distal bowel. To identify key regulators of ENCCs and the pathways underlying RET signaling, gene expression profiles of untreated and GDNF-treated ENCCs from E14.5 mouse embryos were generated. ENCCs express genes that are involved in both early and late neuronal development, whereas GDNF treatment induced neuronal maturation. Predicted regulators of gene expression in ENCCs include the known HSCR genes Ret and Sox10, as well as Bdnf, App and Mapk10. The regulatory overlap and functional interactions between these genes were used to construct a regulatory network that is underlying ENS development and connects to known HSCR genes. In addition, the adenosine receptor A2a (Adora2a) and neuropeptide Y receptor Y2 (Npy2r) were identified as possible regulators of terminal neuronal differentiation in GDNF-treated ENCCs. The human orthologue of Npy2r maps to the HSCR susceptibility locus 4q31.3-q32.3, suggesting a role for NPY2R both in ENS development and in HSCR.

Depletion of the IKBKAP ortholog in zebrafish leads to hirschsprung disease-like phenotype.

AIM: To investigate the role of IKBKAP (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein) in the development of enteric nervous system (ENS) and Hirschsprung disease (HSCR). METHODS: In this study, we injected a morpholino that blocked the translation of ikbkap protein to 1-cell stage zebrafish embryos. The phenotype in the ENS was analysed by antibody staining of the pan-neuronal marker HuC/D followed by enteric neuron counting. The mean numbers of enteric neurons were compared between the morphant and the control. We also studied the expressions of ret and phox2bb, which are involved in ENS development, in the ikbkap morpholino injected embryos by quantitative reverse transcriptase polymerase chain reaction and compared them with the control. RESULTS: We observed aganglionosis (χ2, P<0.01) and a reduced number of enteric neurons (38.8±9.9 vs 50.2±17.3, P<0.05) in the zebrafish embryos injected with ikbkap translation-blocking morpholino (morphant) when compared with the control embryos. Specificity of the morpholino was confirmed by similar results obtained using a second non-overlapping morpholino that blocked the translation of ikbkap. We further studied the morphant by analysing the expression levels of genes involved in ENS development such as ret, phox2bb and sox10, and found that phox2bb, the ortholog of human PHOX2B, was significantly down-regulated (0.51±0.15 vs 1.00±0, P<0.05). Although we also observed a reduction in the expression of ret, the difference was not significant. CONCLUSION: Loss of IKBKAP contributed to HSCR as demonstrated by functional analysis in zebrafish embryos.

A study of calretinin in Hirschsprung pathology, particularly in total colonic aganglionosis.

INTRODUCTION: Calretinin, a calcium-binding protein, has been reported to be an important new marker in Hirschsprung's disease (HD). The aim is to study the diagnostic value of Calretinin in total colonic aganglionosis (TA), prematurity, and superficial biopsy when nerve hyperplasia may not be accessed by ACE activity. METHODS: Records of patients diagnosed with HD at our institution from 1985 to 2010 were studied and patients with TA identified. We examined tissue samples from those TA, partial colectomies for HD, biopsies for suspicion of HD, and rectal tissue from aborted fetuses. Immunohistochemical analysis of Calretinin was compared with ACE gold standard method in all cases. RESULTS: In the majority of the cases, the diagnosis was ascertained by ACE activity and Calretinin staining. However, in 9 cases, the diagnosis was possible with Calretinin staining but not with ACE: in 4 TA because of the absence of nerve hyperplasia, and in 5 cases because the biopsies were too superficial to examine the nerve hyperplasia. In addition, Calretinin was expressed in the gut as early as 22 gestational weeks. CONCLUSION: The use of Calretinin staining may be superior to ACE activity, particularly in the context of TA, superficial biopsies, and prematurity, allowing earlier diagnosis.

Foetal presentation of cartilage hair hypoplasia with extensive granulomatous inflammation.

Cartilage-hair-hypoplasia is a rare autosomal recessive metaphyseal dysplasia due to RMRP (the RNA component of the RNase MRP ribonuclease mitochondrial RNA processing complex) gene mutations. So far, about 100 mutations have been reported in the promoter and the transcribed regions. Clinical characteristics include short-limbed short stature, sparse hair and defective cell-mediated immunity. We report herein the antenatal presentation of a female foetus, in whom CHH was suspected from 23 weeks' gestation, leading to a medical termination of the pregnancy at 34 weeks gestation, and thereafter confirmed by morphological and molecular studies. Post-mortem examination confirmed short stature and limbs, and revealed thymic hypoplasia associated with severe CD4 T-cell immunodeficiency along with extensive non caseating epithelioid granulomas in almost all organs, which to our knowledge has been described only in five cases. Molecular studies evidenced on one allele the most frequently reported founder mutation NR_003051: g.70A>G, which is present in 92% of Finnish patients with Cartilage Hair Hypoplasia. On the second allele, a novel mutation consisting of a 10 nucleotide insertion at position -18 of the promoter region of the RMRP gene (M29916.1:g.726_727insCTCACTACTC) was detected. The founder mutation was inherited from the father, and the novel mutation from the mother. To our knowledge, this case report represents the first detailed foetal analysis described in the literature.

Sox10 and Itgb1 interaction in enteric neural crest cell migration.

SOX10 involvement in syndromic form of Hirschsprung disease (intestinal aganglionosis, HSCR) in humans as well as developmental defects in animal models highlight the importance of this transcription factor in control of the pool of enteric progenitors and their differentiation. Here, we characterized the role of SOX10 in cell migration and its interactions with ß1-integrins. To this end, we crossed the Sox10(lacZ/+) mice with the conditional Ht-PA::Cre; beta1(neo/+) and beta1(fl/fl) mice and compared the phenotype of embryos of different genotypes during enteric nervous system (ENS) development. The Sox10(lacZ/+); Ht-PA::Cre; beta1(neo/fl) double mutant embryos presented with increased intestinal aganglionosis length and more severe neuronal network disorganization compared to single mutants. These defects, detected by E11.5, are not compensated after birth, showing that a coordinated and balanced interaction between these two genes is required for normal ENS development. Use of video-microscopy revealed that defects observed result from reduced migration speed and altered directionality of enteric neural crest cells. Expression of ß1-integrins upon SOX10 overexpression or in Sox10(lacZ/+) mice was also analyzed. The modulation of SOX10 expression altered ß1-integrins, suggesting that SOX10 levels are critical for proper expression and function of this adhesion molecule. Together with previous studies, our results strongly indicate that SOX10 mediates ENCC adhesion and migration, and contribute to the understanding of the molecular and cellular basis of ENS defects observed both in mutant mouse models and in patients carrying SOX10 mutations.

Future therapies for Hirschsprung's disease.

The current management of Hirschsprung's disease (HSCR) is still associated with significant long-term morbidities despite on-going refinements in surgical care. Over the course of the past 20 years, significant inroads have been made in our understanding of the development of the enteric nervous system and what factors are responsible for the development of HSCR. This has prompted increased interest in the possibility of using this knowledge to develop new alternative and adjunctive therapies for HSCR. The aim of this review is to provide an overview of the current progress being made toward the development of future therapies to improve the outcome for children with HSCR.

Skip segment Hirschsprung's disease: a systematic review.

PURPOSE: Hirschsprung's disease is characterised by the congenital absence of ganglion cells beginning in the distal rectum and extending proximally for varying distances. 'Zonal aganglionosis' is a phenomenon involving a zone of aganglionosis occurring within normally innervated intestine. 'Skip segment' Hirschsprung's disease (SSHD) involves a 'skip area' of normally ganglionated intestine, surrounded proximally and distally by aganglionosis. While Hirschsprung's disease is believed to be the result of incomplete craniocaudal migration of neural crest-derived cells, the occurrence of SSHD has no clear embryological explanation. The aim of this study was to perform a systematic review of SSHD, reported in the literature between 1954 and 2009, in order to determine the clinical characteristics of this rare entity and its significance. METHODS: The first reported case of SSHD was published in 1954. A systematic review of SSHD cases in the literature, from 1954 to 2009, was carried out using the electronic database 'Pubmed'. Detailed information was recorded regarding the age, gender, presenting symptoms and location of the skip segment in each patient. RESULTS: 24 cases of SSHD have been reported in the literature to date. 18/24 (75%) of these cases were males and 6/24 (25%) were females. Of these, 22/24 (92%) were cases of total colonic aganglionosis (TCA), and 2/24 (8%) were rectosigmoid Hirschsprung's disease. Of the 22 TCA cases, 9 (41%) had a skip segment in the transverse colon, 6 (27%) in the ascending colon, 2 (9%) in the caecum and 5 (23%) had multiple skip segments. In both rectosigmoid Hirschsprung's disease cases, the skip segment was in the sigmoid colon. Overall, the length of the skip segment was variable, with the entire transverse colon ganglionated in some cases. CONCLUSION: SSHD occurs predominantly in patients with TCA. The existence of a skip area of normally innervated colon in TCA may influence surgical management, enabling surgeons to preserve and use the ganglionated skip area during pull-through operations.

Neural crest regionalisation for enteric nervous system formation: implications for Hirschsprung's disease and stem cell therapy.

Midbrain, hindbrain and vagal neural crest (NC) produced abundant enteric nervous system (ENS) in co-grafted aneural hindgut and midgut, using chick-quail chorio-allantoic membrane grafts, forming complete myenteric and submucosal plexuses. This ability dropped suddenly in cervical and thoracic NC levels, furnishing an incomplete ENS in one or both plexuses. Typically, one plexus was favoured over the other. This deficiency was not caused by lower initial trunk NC number, yet overloading the initial number decreased the deficiency. No qualitative difference in neuronal and glial differentiation between cranial and trunk levels was observed. All levels formed HuC/D+ve, NOS+ve, ChAT+ve, and TH-ve enteric neurons with SoxE+ve, GFAP+ve, and BFABP+ve glial cells. We mathematically modelled a proliferative difference between NC populations, with a plexus preference hierarchy, in the context of intestinal growth. High proliferation achieved an outcome similar to cranial NC, while low proliferation described the trunk NC outcome of incomplete primary plexus and even more deficient secondary plexus. We conclude that cranial NC, relative to trunk NC, has a positionally-determined proliferation advantage favouring ENS formation. This has important implications for proposed NC stem cell therapy for Hirschsprung's disease, since such cells may need to be optimised for positional identity.

Enteric nervous system development: Recent progress and future challenges.

The enteric nervous system is the largest subdivision of the peripheral nervous system that plays a critical role in digestive functions. Despite considerable progress over the last 15 years in understanding the molecular and cellular mechanisms that control the development of the enteric nervous system, several questions remain unanswered. The present review will focus on recent progress on understanding the development of the mammalian enteric nervous system and highlight interesting directions of future research.

Development of the enteric nervous system and its role in intestinal motility during fetal and early postnatal stages.

Motility patterns in the mature intestine require the coordinated interaction of enteric neurons, gastrointestinal smooth muscle, and interstitial cells of Cajal. In Hirschsprung's disease, the aganglionic segment causes functional obstruction, and thus the enteric nervous system (ENS) is essential for gastrointestinal motility after birth. Here we review the development of the ENS. We then focus on motility patterns in the small intestine and colon of fetal mice and larval zebrafish, where recent studies have shown that the first intestinal motility patterns are not neurally mediated. Finally, we review the development of gastrointestinal motility in humans.

Perturbation of hoxb5 signaling in vagal neural crests down-regulates ret leading to intestinal hypoganglionosis in mice.

BACKGROUND & AIMS: The enteric nervous system (ENS) controls intestinal peristalsis, and defective development of this system results in hypo/aganglionosis, as seen in Hirschsprung's disease. In the embryo, vagal neural crest cells (NCC) migrate and colonize the intestine rostrocaudally then differentiate into the ganglia of the ENS. Vagal NCC express the homeobox gene Hoxb5, a transcriptional activator, in human and mouse, so we used transgenic mice to investigate the function of Hoxb5 and the receptor tyrosine kinase gene Ret, which is affected in many patients with Hirschsprung's disease, in ENS development. METHODS: We perturbed the Hoxb5 pathway by expressing a chimeric protein enb5, in which the transcription activation domain of Hoxb5 was replaced with the repressor domain of the Drosophila engrailed protein (en), in vagal NCC. This enb5 transcriptional repressor competes with wild-type Hoxb5 for binding to target genes, exerting a dominant negative effect. RESULTS: We observed that 30.6% +/- 2.3% of NCC expressed enb5 and that these enb5-expressing NCC failed to migrate to the distal intestine. A 34%-37% reduction of ganglia (hypoganglionosis) and slow peristalsis and, occasionally, absence of ganglia and intestinal obstruction were observed in enb5-expressing mice. Ret expression was markedly reduced or absent in NCC and ganglia, and enb5 blocked Hoxb5 induction of Ret in neuroblastoma cells. CONCLUSIONS: Our data indicate that Ret is a downstream target of Hoxb5 whose perturbation causes Ret haploinsufficiency, impaired NCC migration, and hypo/aganglionosis, suggesting that Hoxb5 may contribute to the etiology of Hirschsprung's disease.

[Molecular biology, basic research and diagnosis of Hirschsprung's disease]. / Molekularbiologie, Grundlagenforschung und Diagnose des Morbus Hirschsprung.

The proto-oncogene RET is the major gene responsible for Hirschsprung's disease (HSCR), with RET mutations also implied in different pathologies. A variety of mutations of the RET proto-oncogene have been detected in HSCR patients. Special attention should be paid to rare patients who carry mutations of one of the critical cysteine residues of these exons, known to predispose to MEN2A. In these cases, HSCR can be associated with the development of neuroendocrine tumors such as medullary thyroid carcinoma (MTC) or MEN2A, for which a prophylactic thyroidectomy is advisable in the presence of a tumor causing RET mutation. In combined MEN2A/HSCR families, RET gene testing, tumor screening and prophylactic thyroidectomy are indicated as in MEN2A. The multigenic origin of HSCR and the absence of a "standard" RET mutation associated with HSCR currently make a routine molecular diagnosis impossible.

Reduced endothelin converting enzyme-1 and endothelin-3 mRNA in the developing bowel of male mice may increase expressivity and penetrance of Hirschsprung disease-like distal intestinal aganglionosis.

Hirschsprung disease (distal intestinal aganglionosis, HSCR) is a multigenic disorder with incomplete penetrance, variable expressivity, and a strong male gender bias. Recent studies demonstrated that these genetic patterns arise because gene interactions determine whether enteric nervous system (ENS) precursors successfully proliferate and migrate into the distal bowel. We now demonstrate that male gender bias in the extent of distal intestinal aganglionosis occurs in mice with Ret dominant-negative mutations (RetDN) that mimic human HSCR. We hypothesized that male gender bias could result from reduced expression of a gene already known to be essential for ENS development. Using quantitative real-time polymerase chain reaction (PCR) we demonstrated reduced levels of endothelin converting enzyme-1 and endothelin-3 mRNA in the male mouse bowel at the time that ENS precursors migrate into the colon. Other HSCR-associated genes are expressed at comparable levels in male and female mice. Testosterone and Mullerian inhibiting substance had no deleterious effect on ENS precursor development, but adding EDN3 peptide to E11.5 male RetDN heterozygous mouse gut explants in organ culture significantly increased the rate of ENS precursor migration through the bowel.

The Ret(C620R) mutation affects renal and enteric development in a mouse model of Hirschsprung's disease.

In rare families RET tyrosine kinase receptor substitutions located in exon 10 (especially at positions 609, 618, and 620) can concomitantly cause the MEN 2A (multiple endocrine neoplasia type 2A) or FMTC (familial medullary thyroid carcinoma) cancer syndromes, and Hirschsprung's disease (HSCR). No animal model mimicking the co-existence of the MEN 2 pathology and HSCR is available, and the association of these activating mutations with a developmental defect still represents an unresolved problem. The aim of this work was to investigate the significance of the RET(C620R) substitution in the pathogenesis of both gain- and loss-of-function RET-associated diseases. We report the generation of a line of mice carrying the C620R mutation in the Ret gene. Although Ret(C620R) homozygotes display severe defects in kidney organogenesis and enteric nervous system development leading to perinatal lethality. Ret(C620R) heterozygotes recapitulate features characteristic of HSCR including hypoganglionosis of the gastrointestinal tract. Surprisingly, heterozygotes do not show any defects in the thyroid that might be attributable to a gain-of-function mutation. The Ret(C620R) allele is responsible for HSCR and affects the development of kidneys and the enteric nervous system (ENS). These mice represent an interesting model for studying new therapeutic approaches for the treatment of HSCR disease.

Location of stem cells for the enteric nervous system.

Hirschsprung disease is the result of aganglionosis of a variable length of the terminal bowel, which arises from the incomplete colonisation of the embryonic gut by vagal neural crest-derived cells (NCC) that migrate caudally from the pharyngeal gut to the rectum. We have previously shown that a very small group of NCC, at the leading edge of this wave of migration, can proliferate and differentiate to innervate the entire distal gut. It remains unknown if this capability is unique to those cells at the leading edge of NCC migration. The hypothesis tested was that NCC capable of acting as stem cells are found throughout the developing enteric nervous system (ENS). Gut was taken from mice at embryonic day 11.5 as the leading edge of NCC migration enters the colon. Terminal colon was separated as aganglionic recipient gut and its rostral end juxtaposed to the caudal end of the small intestine or caecum. The explants were cultured on nitrocellulose filters for up to 120 h, after which time the apposed segments had fused. The gut was then fixed and examined by immunohistochemistry to detect the neuronal markers PGP9.5 and nitric-oxide synthase (NOS) to assess development of enteric ganglia. NCC migrated from the proximal gut into the terminal colon, colonising it along its entire length. The pattern of NCC colonisation and differentiation of NOS-positive neurons was the same, regardless of whether the NCC were derived from the leading edge of migration in the caecum or from more proximal regions of the small intestine. Vagal NCC have the capacity to migrate into separated aganglionic terminal colon and differentiate into neurons. NCC at the leading edge of migration and those located more proximally within the gut demonstrate equivalent ability to migrate to and differentiate in the terminal rectum. Further studies are required to confirm which of these migrating NCC have the properties of ENS stem cells.

Enteric nervous system: development and developmental disturbances--part 1.

This review, which is presented in two parts, summarizes and synthesizes current views on the genetic, molecular, and cell biological underpinnings of the early embryonic phases of enteric nervous system (ENS) formation and its defects. In the first part, we describe the critical features of two principal abnormalities of ENS development: Hirschsprung's disease (HSCR) and intestinal neuronal dysplasia type B (INDB) in humans, and the similar abnormalities in animals. These represent the extremes of the diagnostic spectrum: HSCR has agreed and unequivocal diagnostic criteria, whereas the diagnosis and even existence of INDB as a clinical entity is highly controversial. The difficulties in diagnosis and treatment of both these conditions are discussed. We then review the genes now known which, when mutated or deleted, may cause defects of ENS development. Many of these genetic abnormalities in animal models give a phenotype similar or identical to HSCR, and were discovered by studies of humans and of mouse mutants with similar defects. The most important of these genes are those coding for molecules in the GDNF intercellular signaling system, and those coding for molecules in the ET-3 signaling system. However, a range of other genes for different signaling systems and for transcription factors also disturb ENS formation when they are deleted or mutated. In addition, a large proportion of HSCR cases have not been ascribed to the currently known genes, suggesting that additional genes for ENS development await discovery.

Enteric nervous system: development and developmental disturbances--part 2.

This review, which is presented in two parts, summarizes and synthesizes current views on the genetic, molecular, and cell biological underpinnings of the early embryonic phases of enteric nervous system (ENS) formation and its defects. Accurate descriptions of the phenotype of ENS dysplasias, and knowledge of genes which, when mutated, give rise to the disorders (see Part 1 in the previous issue of this journal), are not sufficient to give a real understanding of how these abnormalities arise. The often indirect link between genotype and phenotype must be sought in the early embryonic development of the ENS. Therefore, in this, the second part, we provide a description of the development of the ENS, concentrating mainly on the origin of the ENS precursor cells and on the cell migration by which they become distributed throughout the gastrointestinal tract. This section also includes experimental evidence on the controls of ENS formation derived from classic embryological, cell culture, and molecular genetic approaches. In addition, for reasons of completeness, we also briefly describe the origins of the interstitial cells of Cajal, a cell population closely related anatomically and functionally to the ENS. Finally, a brief sketch is presented of current notions on the developmental processes between the genes and the morphogenesis of the ENS, and of the means by which the known genetic abnormalities might result in the ENS phenotype observed in Hirschsprung's disease.

Localization and endothelin-3 dependence of stem cells of the enteric nervous system in the embryonic colon.

BACKGROUND/PURPOSE: The aganglionosis in a variable length of the distal gut found in Hirschsprung's disease results from the abnormal prenatal development of neural crest-derived stem cells of the enteric nervous system. The cytokine endothelin-3 is necessary for successful colonization of the distal gut, but the location of this interaction with neural crest-derived stem cells remains to be established. The hypothesis tested here is that the stem cells of the enteric nervous system (ENS) in the colon are located at the leading edge of the migrating wave of neural crest-derived stem cells and that these cells require colonic endothelin-3 for complete colonization of the gut. METHODS: Explants of 11.5-day-old embryonic intact mouse gut and isolated colon were cultured for 72 hours in the presence and absence of the endothelin-B receptor antagonist, BQ788. Specimens then were sectioned and stained by immunohistochemistry to assess enteric nervous system development. RESULTS: Isolated colon contained a very low number (mean, 73 cells; range, 37 to 106; n = 8) of neural crest-derived stem cells, which had just entered its proximal end at the leading edge of neural crest cell migration. After 72 hours of culture, progeny of these few neural crest-derived stem cells had colonized the colon at an equivalent ganglionic density to those in intact gut. Furthermore, neuronal differentiation, as shown by the appearance of nitric oxide synthase positive neurons, also was equivalent to intact gut. Blockade of the endothelin-B receptor produced terminal aganglionosis in both isolated colons and intact gut. CONCLUSIONS: The very small number of cells that first enter the proximal colon at the leading edge of neural crest cell migration have the ability to colonize the entire colon normally in an ET-3-dependent manner. These cells therefore have the functional characteristics expected of the stem cells of the colonic enteric nervous system. Furthermore, the normal development of these cells is dependent on the endothelin-3 expressed by the mesenchymal cells of the colon itself.